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IDENTIFICATION AND CHARACTERIZATION OF A MAJOR HEPATIC GLUTATHIONE S -TRANSFASE ISOENZYME IN LARGEMOUTH BASS ( Micropterus s almoides ) THAT CONJUGATES 4-HYDROXYNON-2-ENAL. BY ROBERT TRAN PHAM A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMEN T OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2003

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Copyright 2003 by Robert Tran Pham

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ACKNOWLEDGEMENTS I would like to thank my committee chair, Dr. Evan P. Gallagher, and my committee members Dr. David S. Barber and Dr. Nancy Denslow. I would to express my appreciaton to the Gallagher group: Max Huidsen, Erin Hughes, Kathy Childress, and Dr. Craig Moneypenny. Finally, I would like to thank Dr. Adrianna Doi and Dr. James Gardner. This thesis would not have been possible without their support and advice. iii

Abstract of Thesis Presented to the Graduate School of The University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science IDENTIFICATION AND CHARACTERIZATION OF A MAJOR HEPATIC GLUTATHIONE S-TRANSFASE ISOENZYME IN LARGEMOUTH BASS (Micropterus salmoides) THAT CONJUGATES 4-HYDROXYNON-2-ENAL. By Robert Tran Pham December 2003 Chair: Evan P. Gallagher Major Department: Veterinary Medicine The glutathione S-transferases (GST) are a multigenic family of phase II enzymes involved in the detoxification of carcinogenic and reactive intermediates. Certain GST isozymes, including those of the mammalian alpha class, have particularly high activity toward alkenals including 4-hydroxynon-2-enal (4HNE), and other reactive by-products produced during lipid peroxidation. The ability of cells to remove 4HNE is of particular importance since 4HNE is an extremely carcinogenic and mutagenic intermediate produced at relatively high concentrations on exposure to peroxidizing chemicals. In general, relatively little is known regarding the ability of fish species to detoxify 4HNE and related products of oxidative injury via GST. In this thesis, we ix

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have identified and characterized the GST isoform responsible for the rapid metabolism of 4HNE in largemouth bass, a freshwater species and higher order predatory species that tends bioaccumulate lipophilic toxicants through the food chain. HPLC-GST subunit analysis revealed the presence of at least two major GST isoforms in bass liver, with the first peak (peak one) constituting 80% of the total bass liver GST protein. HPLC with electrospray-ionization of the two isolated GST subunits yielded molecular weights of 26,396 kDa and 25,515 kDa. Endo-proteinase Lys-C digestion and Edman degradation protein sequencing of this GST isoform that is similar to the plaice GST isoform that rapidly metabolizes 4HNE was termed bass GSTA. Peak one demonstrated that this major GST isoform was encoded by GSTA. Analysis of genomic DNA fragments isolated by nested PCR indicated the presence of a GST gene cluster in bass liver that is similar to plaice GST gene cluster. Using nested deletions with Exonuclease III and Mung Bean nuclease, we were able sequence the entire upstream bass GSTA promoter. Isolation of approximately 1 kb of the bass GSTA promoter revealed the presence of several putative response elements that may confer inducibility to endogenous and environmental chemicals. Collectively, our data indicates the presence of a major GST in bass liver involved in the protection against oxidative stress. Furthermore, this GST is part of a gene cluster that may be conserved in aquatic species. x

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CHAPTER 1 INTRODUCTION Biotransformation Pathways of Detoxification All organisms are constantly exposed to a variety of foreign chemicals, which include synthetic and natural chemicals such as chemotherapeutic drugs, industrial chemicals, pesticides, polycyclic aromatic hydrocarbons (PAH), and natural toxins from plants, molds, fungus and animals. The physical property that allows many xenobiotics to be absorbed through the skin, lungs, gastrointestinal tract, and other various parts of the body is lipophilicity. Consequently, the elimination of xenobiotics depends on the conversion of xenobiotics to water-soluble compounds by a process known as biotransformation, which is catalyzed by biotransformation enzymes in the liver and other tissues (Klaasen, 2001). The reactions catalyzed by xenobiotic biotransformation enzymes are grouped in two categories: phase I and phase II enzymes. Phase I biotransformation reactions involve hydrolysis, reduction, and oxidation reactions and results in the addition or exposure of functional groups (e.g -OH, -NH 2 -SH or -COOH). The phase I enzymes include aldehyde dehydrogenase, flavin monooxygenase, and cytochrome P450s. However, some phase I biotransformation pathways does not always lead to detoxification reactions. For example, aflatoxin B 1 (AFB 1 ), a known natural occurring hepatocarcinogen produced by the mold Aspergillus flavus, is bioactiviated by cytochrome P4501A2 (CYP1A2 isoform) to an ultimate 1

3 androsten-3,17-dione (ADI, selective specificity with class rat rGSTA1 and rGSTA2), 4-hydroxynonenal (4HNE, selective for class rat rGSTA4 and human hGSTA4), and 1,2-epoxy-3-p-nitrophenoxy propane (EPNP, selective for class rat rGSTT1) (Hayes and Pulford, 1995). Other GST substrates include carcinogens (AFB 1 -8-9-epoxide), pesticides (DDT, atrazine), anti-cancer drugs (BCNU, chlorambucil) and by-products of lipid peroxidation (fatty acid hydroperoxides, ,-unsaturated aldehydes) (Hayes and Pulford, 1995). The mechanism of GST-mediated conjugation typically involves electrophilic conjugation with the tripeptide glutathione. The dimeric GST subunit has an active site composed of 2 distinct regions: G-site (hydrophilic binding site of substrate GSH) and an adjacent H-site (active site for variety of electrophilic substrates) (Mannervik and Danielson, 1988; Armstrong, 1997). Thus, the G-site is conserved in all GST families due to its high affinity for GSH, while the H-site shows a broad range of electrophilic substrate binding affinities can differ between GST families (Hayes and Pulford, 1995). GST catalyzes the general reaction shown: GSH + R-X -----GST----> GSR + HX The catalytic reaction of GST involves positioning the substrate within close proximity of GSH for binding of GSH and the electrophilic substrate to the active site of the protein, and activating the sulfhydryl group on GSH, thereby allowing a nucleophilic attack of GSH on any electrophilic center of the substrate (R-X) (Armstrong, 1997). The GSH conjugates formed in the liver can be excreted in bile, or can be converted to mercapturic acids in the kidney and excreted in urine.

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4 The conversion of GSH conjugates to mercapturic acids involves sequential cleavage of glutamic acid and glycine (by -glutamlytranspeptidase and aminopeptidase M, respectively) from the GSH conjugate, followed by N-acetylation of the cysteine conjugate (Klaasen, 2001). Besides having catalytic activity towards reactive intermediates, certain GST isoenzymes have non-catalytic properties such as intracellular carrier proteins for steroid and thyroid hormones, bile acids, bilirubins, and fatty acids (Hayes and Pulford, 1995). Further, some GST isoenzymes show a glutathione-peroxidase-like activity (general reaction: ROOH + GSH -----GST----> ROH + GSSG + H 2 O) in which organic peroxides are converted to the corresponding alcohol (Hayes and Pulford, 1995). The expression of certain cytosolic GST isoforms can be induced by exposure to certain xenobiotics, including PAH, reactive oxygen species (ROS), Michael acceptors, phenolic antioxidants, and glucocorticoids (Hayes and Pulford, 1995). Induction of GST can involve several transcriptional mechanisms. For example, the induction of mammalian GSTs by xenobiotics can be mediated by several sequence-specific DNA motifs (xenobiotic response element (XRE), anti-oxidant response element (ARE), glucocorticoid response element (GRE), located in the regulatory regions of all genes and which respond to intracellular or extracellular stimulus by activating the transcription factors that bind to the motif and that either upor down regulate gene expression (Dynan and Tjian, 1985). For example, the rat rGSTA2 gene contains a xenobiotic response element (XRE: TA/TGCGTG), an anti-oxidant response element (ARE:

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5 TGACAAAAGC), and a glucocorticoid response element (GRE: AGAACANNNTGTTCT) (Hayes and Pulford, 1995). The XRE facilitates induction by various compounds such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), while the GRE mediates induction by synthetic glucocorticoids such as dexamethasone. Phenolic antioxidants such as tert-butlyhydroquinone (TBHQ) can induce GST via ARE consensus sequences (Hayes and Pulford, 1995). In mice, mGSTA2 contains a response element related to the ARE called the electrophile response element (EPRE: TGACNNNNGC) (Hayes and Pulford, 1995). The EPRE contains two tandem arrangements of consensus sequence ARE elements that confer induction in response to certain chemicals such as -naphthoflavone (Hayes and Pulford, 1995). Interestingly, AREs and EPREs have similar consensus sequences (TGACNNNNGC) which are often referred as AP1-like binding sites. Thus, it is proposed that a variety of chemical agents PAH, diphenols, phenobarbital and electrophilic compounds can induce mouse GST (mGSTA1) by the activation of the Fos/Jun heterodimeric complex (AP1) (Bergelson et al., 1994; Hayes and Pulford, 1995). Piscine Glutathione S-Transferases Although mammalian GSTs have been extensively characterized, much less is known about fish GST isoenzymes. As in mammals, fish GSTs can conjugate various electrophilic environmental chemicals (George, 1994). Proteins related to mammalian alpha (), mu (), pi () and theta () class GSTs have been described in various fish species (George, 1994). In particular, the

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6 structure and expression of three theta-like GST genes (GSTA, GSTA1, and the pseudogene, GSTA2) from the plaice (Pleuronectes platessa) have been characterized and identified (Leaver et al., 1997). Analysis of the promoter of plaice GSTA1 gene revealed multiple peroxisome proliferator response elements (PPRE) similar to murine PPRE (Zimniak et al., 1994). The plaice GSTA and GSTA1 gene has been found to be up-regulated after administration of perfluoro-octanoic acid (PFOA), a potent peroxisome proliferators, while only the GSTA gene was induced by -naphthoflavone (BNF), a classic bifunctional inducer of phase I and II biotransformation enzymes (Leaver et al., 1997). This suggests that the PPRE and ARE in plaice are regulated in a similar manner to that described for mammalian genes. The presence of a transposon-like element (PPTN) has been identified between the GSTA1 and GSTA gene cluster, and the PPTN contains multiple AREs (Leaver et al., 1997). Also, the presence of an estrogen response element (ERE) in the plaice GSTA1 promoter may suggests a possible role for increasing GST-mediated detoxification of lipid peroxides during reproduction, as fish lipid membranes consist heavily of polyunsatured fatty acids (Hyllner et al., 1994). In additions to the studies in plaice, Carvan et al. have developed a zebrafish transgenic model system in which DNA motifs (XRE, ARE, and EPRE) that respond to environmental pollutants by activating a reporter gene (2000). Other studies have shown that hepatic GSTs can be induced in brown bullhead and channel catfish by electrophilic agents or anti-oxidant agents (Gallagher et al., 1991; Henson et al., 2001). Thus, it appears that many fish

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7 GST isoenzymes exhibit similar induction mechanisms observed for mammalian GSTs. Role of GST in Protecting Against Oxidative Injury Cellular respiration or oxidative process produces several reactive free radical intermediates which are the initiating factors in the decomposition of lipids, and ultimately producing ,-unsaturated aldehydes. In this regard, the superoxide anion radical (O 2 ), hydroxide radical (OH), and hydrogen peroxide (H 2 O 2 ) constitute primary reactive oxygen species (ROS) (Halliwell and Gutteridge, 1999). ROS can attack DNA, proteins, and cellular targets such as polyunsaturated fatty acids (PUFA). The process of lipid peroxidation is initiated by a hydroxy radical OH (Figure 1). The lipid radical (L) is converted to lipid peroxyl radical (LOO) by an addition of oxygen, lipid hydroperoxide (LOOH) by hydrogen abstraction, and lipid alkoxyl radical (LO) by the Fe +2 -catalyzed Fenten reaction (Figure 1). The end-products of lipid peroxidation are reactive aldehydes such as malondialdehyde (MDA) and 4-hdyroxynonenal (4HNE). Certain GST isoforms have conjugative activity towards endogenous genotoxic -unsaturated aldehydes formed during lipid peroxidation (Mannervik and Danielson, 1988). In this regard, the GST detoxification pathways play an important role in protection against reactive oxygen species and their electrophilic reactive intermediates. In particular, 4HNE is the end product of arachidonic acid peroxidation, and is an extremely genotoxic, mutagenic and long-lived compound and can readily react with adjacent molecules (proteins and lipids) or diffuse to distant targets such as DNA (Esterbauer et al., 1991).

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8 Figure 1. A brief schematic showing the initiation of lipid peroxidation by hydroxyl radical (OH) and its end products are ,-unsaturated aldehydes (e.g 4HNE, MDA). The effects of 4HNE depend upon the concentration of 4HNE. At cellular levels of 100 nm or lower, 4HNE can stimulate chemotaxis and phospholipase C (Eckl et al., 1993). At levels of 1-20 M, DNA and protein synthesis are inhibited, there is an increase chromosomal aberrations and sister chromatid exchange, and cell proliferation is inhibited (Eckl et al., 1993). Levels of 100 M or above may

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9 occur near peroxidizing membranes and can cause cell lysis and cell death (Eckl et al., 1993; Halliwell and Gutteridge, 1999). Coincidently, elevated tissue 4HNE concentrations have been associated with several human diseases, including cancer (Eckl et al., 1993), Parkinsons disease, Alzheimers disease (Markesbery and Lovell, 1998), atherosclerosis (Chen et al., 1995; Muller et al., 1996), pulmonary inflammation (Hamilton et al., 1996), rheumatoid arthritis (Selley et al., 1992), ophthalmologic disorders (Esterbauer et al., 1991), and liver disease (Tsukamoto and French, 1993). Given the high reactivity and toxicological importance of 4HNE, it is not surprising that a number of enzyme systems have evolved to protect tissues from 4HNE injury (Esterbauer et al., 1991). The primary enzymatic pathways of 4HNE detoxification in adult human liver include aldehyde dehydrogenase (ALDH), alcohol dehydrogenase (ADH), aldehyde reductase (ALRD), and GST (Figure 2) (Mitchell and Petersen, 1987; Sellin et al., 1991; Hayes and Pulford, 1995). Goals of the Present Study We have previously described the in vitro kinetics of GST-CDNB conjugation in largemouth bass, a freshwater fish and a higher order predatory species that has been shown to bioaccumulate hydrophobic xenobiotics and is sensitive to the toxic effects of environmental contaminants (Gallagher et al., 2000). Furthermore, we have cloned and expressed a recombinant bass GSTA protein that has high catalytic activity towards 4HNE (Doi et al., 2003). This bass GSTA exhibits high homology to the plaice GSTA that also conjugates 4HNE.

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10 Figure 2. Enzymatic pathways of detoxification of 4HNE involve reduction via aldehyde reductase or alcohol dehydrogenase, oxidation via aldehyde dehydrogenase or conjugation with GSH via GST. What is not known is: 1) the identity of the bass GST isoenzyme(s) involved in the high metabolism of 4HNE, 2) the enzymatic and immunological characteristics of the bass GST isoenzymes(s), and 3) genomic information on the GST gene, and specifically, the presence of regulatory elements that may confer induction by environmental compounds. Accordingly, the specific aims and hypothesizes are the following: Specific Aim 1: Fully characterize GST isoenzyme mediated 4HNE conjugation in bass liver. Hypothesis: High efficiency single-enzyme Michaelis-Menten kinetics of GST-4HNE conjugation is observed in bass liver, suggesting that a single GST isoenzyme is responsible for 4HNE metabolism.

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11 Specific Aim 2: Determine the number of major GST isoenzyme(s) in bass liver cytosol and determine if GSTA encodes a highly expressed cytsolic GST. Hypothesis: Bass express multiple GST subunits, however GSTA is a major GST isoform in bass liver. Specific Aim 3: Obtain at least a 1kb 5 flanking region of the GSTA promoter that encodes a protein that is involved in GST-4HNE conjugation. Analyze for the presence of regulatory elements that may potentially confer changes in gene expression in response to environmental chemicals. Hypothesis: The 5 flanking region of bass GSTA gene contains several classes of regulatory elements homologous to mammalian response elements (ARE, XRE, ERE, NF-B, EPRE and GRE) that modulate gene transcription on exposure to environmental agents.

13 endonucleases were purchase from New England Biolabs (Beverly, MA). All PCR primers were synthesized by IDT DNA (Skokie, IL). The Exonuclease III / Mung Bean Deletion kit was provided by Stratagene (La Jolla, CA.). Animals Largemouth bass (LMB) were collected from Lake Woodruff, a non-polluted site located on a National Wildlife Refuge. Hepatic cytosolic fractions were prepared from adult, reproductively inactive (mixed sexes, aged 2-5 years) (Guillette et al., 1994; Gallagher et al., 2000). In addition, for some studies, aquacultured juvenile largemouth bass (200-300g) were obtained from American Sportfish Hatchery (Montgomery, Ala.). The fish were sacrificed by a blow to the head, and the brain, heart, liver, lower and upper gastrointestinal tracts, gills, and muscles were extracted, snap frozen in liquid N 2 and stored at -80 o C. Hepatic cytosolic fractions from brown bullheads, Sprague-Dawley rat, and adult human tissues were available from previous studies in our laboratory. Subcellular Fractions Affinity purification of bass liver GST Hepatic liver cytosolic fractions were prepared as previously described (Gallagher et al. 2000) by differential centrifugation. Cytosolic fractions were prepared by using a MicroSpin TM spin column and GSH Sepharose 4B matrix according to manufacturer's directions (AmershamPharmacia, Piscataway, NJ). For affinity purification, approximately 1.2 ml of liver cytosol was equilibrated with PBS, and 400 l of the liver cytosolic fractions containing 5 mM DTT and 1.0 mM PMSF were applied to the purification columns. The columns were mixed gently at room temperature for 10 min, followed by centrifugation at 400 x g for 1 min.

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14 The columns were then washed twice with PBS and centrifuged at 400 x g for 1 min, followed by elution of GST proteins with 200 l of GSH elution buffer (GEB, 10 mM Tris-HCl, 1.4 mM BME, 150 mM reduced glutathione, pH 9.6). A second elution was performed with 100 l of GEB and the eluates from each step were pooled. Eluates were dialyzed for 48 hr, with a change of fresh PBS every 6 hr, using a QuixSep TM micro-dialyzer system (Membrane Filtration Products, San Antonio, TX).) and Spectro-Pro membrane (MWCO 3.5 kDa, Spectrum Laboratories, IN). Protein concentrations of the affinity-purified samples were determined by the bicinchoninic-acid assay with bovine serum albumin as the standard (Smith et al., 1985). RNA isolations Total RNA isolation from bass liver was achieved by a modified method of Chomczynski and Sacchi (1987), using the Trizol solution reagent (Invitrogen, Carlsbad, CA). Approximately 300 mg of liver was homogenized in 3 ml of Trizol and incubated at RT for 5 minutes. Approximately 200 l of chloroform was added then transfer to a new 1.5 mL tube then equal volume of Trizol solution was added (approximately 500 l) and incubated at room temperature for 5 minutes. An additional 500 l of Trizol was used to further facilitate removal of proteins and other superfluous cellular materials that were not completely isolated from the first round of Trizol. The final solution was centrifuged at 12,000 g at 4C for 15 minutes. Approximately 500 l of isopropanol was added to the aqueous phase and subjected to another round of centrifugation. The supernatant was discarded and the pellet was washed in 75% EtOH. The purified RNA was resuspended in nuclease-free H 2 0 (Gibco/Invitrogen, Carlsbad,

17 modifications. Reverse-phase HPLC was used to characterize the GST subunit composition using a 150 x 4.6 mm Vydac C4 column (Grace Vydac, Hesperia, CA). Samples (approximately 60 g) were mixed with equal volume of 0.075% trifluoroacetic acid (TFA) and injected onto the column attached to a Perkin Elmer 200 series HPLC system. The HPLC system was equilibrated with 37% (v/v) acetonitrile in 0.075% TFA. The column flow rate was 1.5 ml/min with 37-43% (v/v) gradient of acetonitrile containing 0.075% TFA over 25 minutes. This was followed by a linear increase of 43-55% (v/v) gradient of acetonitrile containing 0.075% TFA between 25-45 minutes. Polypeptide peaks were detected with a diode array detector monitoring absorbance at 214 nm. Peak area integrations were performed using Perkin Elmer Turbochrom Software. The HPLC fractions (polypeptide peaks) were collected by hand and the polypeptides were dried under reduced-pressure in a Speed-Vac centrifuge overnight to remove the TFA. HPLC Mass Spectrometry with Electrospray-ionization Analysis The molecular weights of the affinity-purified bass hepatic GST proteins were determined by HPLC mass spectrometry (HPLC-MS) with electrospray-ionization (ESI). The GST proteins (approximately 60 g) were dissolved in equal volume of water containing 0.075% trifluoroacetic acid and injected at a rate of 1.5 ml/min into the ESI ion source. Positive ion ESI-mass spectra were acquired using a Thermo-Finnigan LCQ-Classic ion trap mass spectrometer. The ESI source was operated at 4.2 kV with the heated capillary at 220C and a relative nitrogen flow of 80%. Spectra were scanned from m/z 200-2000 and

19 sec, 56 o C for 1 min, 72 o C for 30 sec) and final extension of 72 o C for 5 minutes. The PCR products were separated on 2% ethidium bromide agarose gel and visualized using a Flour-S Multimager system (BioRad, Hercules, CA). Sequence Analysis of the GSTA Gene Initial characterization of bass liver genomic sequences The Universal GenomeWalker kit (Clonetech Inc., Palo Alto, CA) was used to isolate the 5 flanking region and other genomic sequences of the bass GSTA gene. Genomic DNA was isolated from snap frozen bass liver using the Wizard genomic DNA purification kit (Promega Corp., Madison, WI). Bass genomic DNA was subjected to an overnight digestion (to ensure complete digestion) with the following blunt-end endonuclease enzymes: Dra I, EcoR V, Pvu II, and Stu I. Following the overnight digestion, each restriction-digested bass genomic DNA fragment was subjected to a phenol / chloroform extraction to remove proteins and restriction enzymes. An additional round of chloroform extraction was used to remove residual phenol. Each batch of digested genomic DNA fragment ends was ligated into a GenomeWalker adaptor (provided in the kit) to make GenomeWalker libraries. The GenomeWalker adaptor is a 52-mer oligonucleotide that has complementary sites for the forward adaptor primers (AP1 and AP2, provided in kit) to be used in primary and nested PCR reactions. The GenomeWalker adaptor has three design features that are critical to the success of the PCR reaction: 1) the use of a 5 extended adaptor has no binding site for adaptor primer 1 used in primary PCR, 2) the addition of amino group at the 3 end to prevent primer dimerization with the adaptor primer, and 3) the use of a adaptor primer that is shorter than the adaptor itself would cause

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20 suppression PCR. The GenomeWalker DNA walking requires eight primary and secondary (nested) PCR amplications: four experimental genomic DNA libraries, two positive controls (positive control with human pre-constructed library, and the second positive control library from control human genomic DNA), and two negative controls (no genomic library templates were used in the PCR reactions) (Figure 3). Figure 3. Flowchart of the GenomeWalker protocol for the isolation of GST genomic clones.

22 The gene-specific primers were designed by using Oligo Software (Molecular Biology Insights, Cascade, CO.). The nested PCR products were visualized on a 1.5% ethidium bromide agarose gel and purified by a gel extraction kit (Qiagen, Valencia, CA). The purified PCR products were cloned into pGEM T-easy vector (Promega Corp., Madison, WI) and submitted for nucleotide sequencing to University of Florida Interdisciplinary Center for Biotechnology Research (ICBR) DNA Sequencing Core (Gainesville, FL). Upon receiving sequence information, ClustalW software was used to align the sequenced fragments with the plaice GST gene cluster, and the BLAST nucleotide search engine was used to identify the sequence fragments. Nested deletion analysis of GST genomic clones Exonuclease III and the Mung Bean Deletion Kit were used to make unidirectional nested deletions on clones derived from GenomeWalker analysis (Stratagene, La Jolla, CA). One of the criteria was to select the appropriate restriction endonucleases to linearize the pGEM T-easy vector plasmid. Exonuclease III will progressively digest the 3 end of double-stranded DNA or blunt ends, but can not efficiently initiate digestion at a 3 overhang end or a 5 overhang end that is filled-in with -thio dNTPs. To create deletions in the insert but not in the vector, the plasmids of interest were linearized by a double-digestion with a 3-overhang restriction endonuclease and a 5-overhang restriction endonuclease to create a substrate for unidirectional exonuclease digestion by Exonuclease III. According to the reference restriction sites of the pGEM T-easy vector, there are several unique restriction sites that can used on both sides of the inserted DNA (Figure 4). Since several 3 overhang restriction

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23 enzymes digested the insert of the plasmid, two 5-overhang restriction enzymes were selected: Nde I and Spe I. Figure 4. The promoter and multiple cloning sequence site of the pGEM T-easy vectors. Since two 5-overhang restriction endonucleases were used on the same side of the insert, a thioderivative fill-in with Klenow fragment was required to protect one of the sites from Exonuclease III digestion. Approximately 25 g of clone 2F (5.5 kb inserted DNA) was digested in 500 l reaction with Nde I for 3 hr at 37C. After the 2F clone was completely digested, a 5 overhang fill-in reaction was performed using 1mM thio-dNTP mix and 5 U of Klenow fragment and incubated at room temperature for 10 minutes. After the fill-in reaction, the reaction was extracted using a phenol / chloroform and EtOH to remove residual restriction enzymes. Verification of the thioderivative filled-in reaction was achieved by incubating 1 g of filled-in DNA with 20 U of Exonuclease III for 15 minutes at 37C and visualization using a 1% ethidium bromide agarose gel. After the first digestion, the filled-in DNA was subjected to a second round of 5

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24 overhang restriction digest using Spe I and another round of a phenol / chloroform and EtOH extractions as described above. The length of restriction digested DNA converted from double-stranded to single-stranded by Exonuclease III is controlled by the reaction temperature and time of incubation. At 23C, Exonuclease III can digest approximately 500 base pairs per 4 minutes. Accordingly, ten time points (500 bp x 10 = 5.0 kbp) were selected for analysis with each time point reaction consisting of 5.0 g of double-digested DNA, 2X Exo III buffer, and 100 mM BME. Each time point reaction was initiated by 100 U of Exonuclease III. At every 4 minutes, aliquots were removed and heated to 68C for an additional 15 minutes. Mung bean nucleases (45 U) were added to each time point reaction and incubated at 37C for an additional 30 minutes. Prior to ligation, a modified version of the phenol / chloroform extraction procedure which includes 1m Tris-HCL, 8M Li-Cl, and 20% SDS was used to remove any residual Mung Bean nucleases. The Exonuclease III / Mung Bean nuclease-treated DNA was subjected to overnight ligation at 4C and transformed into JM109 cells following the protocol from pGEM T-easy vector system (Promega, Madison, WI). Blue-white colonies were screened and plasmids from each time point reactions were purified using Wizard mini-prep kit (Promega, Madison, WI). The purified plasmids were sequenced at the University of Florida DNA Sequencing Core.

CHAPTER 4 DISCUSSION Although much less is known about piscine GSTs relative to mammalian species, previous studies have shown that various fish species can conjugate a broad range of electrophilic substrates (CDNB, NBC, ECA and ADI) via GST, and that GST proteins related to mammalian , and class have been described in several aquatic species (George, 1994). In particular, a previous study in our laboratory showed that bass hepatic GST cytosolic fractions rapidly metabolized 4HNE (Pham et al., 2002), however the identity of the bass GST isoenzyme(s) responsible for the high 4HNE activity remained elusive. In the present study, we observed that the initial rates of GST-4HNE activities were relatively rapid in bass compared to other species. Furthermore, the very high GST-4HNE / GST-CDNB activity ratios indicated that a high proportion of the total GST cytosolic protein in bass is dedicated to the metabolism of 4HNE. Given that largemouth bass are higher order predators whose diet consists of lipid materials and whose lipid membranes are rich in polyunsaturated fatty acids, it likely that the high level of GST-4HNE activity functions to protect against oxidative injury. The two-enzyme Michaelis-Menten curves of the GST-CDNB activity data suggests the presence of multiple bass liver GST isoenzymes each with different affinity for CDNB. These observations were supported by the presence of the two GST isoenzymes by our SDS-PAGE analysis. This data is also consistent 38

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39 with a previous enzyme kinetics study by Gallagher et al. (2000). However, the enzyme kinetics of GST-4HNE reaction data revealed a linear relationship among substrate concentration and reaction velocity suggests the presence of single GST isoenyzme responsible for 4HNE metabolism. In addition, the apparent K m and apparent V max values (18.9 + 1.3 M and 24 + 0.5 mol 4HNE conjugated/min/mg, respectively) suggest that this GST isoenzyme has very high affinity and high catalytic efficiency for catalyzing 4HNE conjugation. Interestingly, the recombinant plaice GSTA also exhibits relatively high efficiency towards 4HNE, with Km of 150 85.0 uM and a Vmax of 3.6 1.8 umol 4HNE conjugated/min/mg (Leaver and George, 1998). Based upon the immunoblotting studies, it appears that bass cytosolic GST isoenyzme(s) share little identity with the better-characterized rodent alpha, mu, pi, and theta class GSTs. This data is also supportive of the presence of one or more novel bass liver GSTs compare to rodent GSTs. Similarly, English sole and Starry flounder cytosolic GSTs exhibit high activity toward class-specific GST substrates, but do not show any strong cross-reactivity using rodent class-specific GST antibodies (Gallagher et al., 1998). In most fish species, it appears that a discordance exists using class-specific substrates and antibodies directed against the rodent GSTs. Accordingly, it is evident that care should be taken for predicting or identifying the presence of GST classes in fish based upon the use of mammalian GST probes. Our kinetics data of GST-4HNE conjugation using GSH affinity-purified cytosolic fractions suggests that a single GST isoenyzme is responsible for the

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40 4HNE conjugating activity in bass liver. Accordingly, reverse phased HPLC analysis was used to characterize the GST subunit composition of the GSH affinity purified fractions. At least 2 peaks were eluted, the major peak (peak 1) constituted approximately 80% of the total cytosolic GST protein, assuming that the extinction coefficients were similar for the GST subunits. Furthermore, sequence analysis of peak 1 revealed a 14 amino acid sequence with high identity to the recombinant GSTA protein that was cloned by Dr. Doi et. al. and exhibiting a molecular weight of 26.3 kDa (2003). Therefore, the major peak (peak 1) appears to be GSTA and is likely the major GST isoenyzme in bass liver. In addition to liver, bass GSTA mRNA was also present in gonad, upper gastrointestinal tract, and brain tissue. However, no detectable expression of GSTA mRNA was observed in heart, lower gastrointestinal tract or muscle tissue. This is in contrast to the studies of Leaver et. al. who demonstrated that plaice GST-A mRNA was expressed in all tissues including liver, intestine, gill, kidney, brain, gonad, heart, spleen and testis (1997). Leaver et. al. proposed that the expression of plaice GSTA mRNA in all tissues indicated a housekeeping function for the GSTA gene (1997). However, the differences in tissue expression of bass GSTA mRNA and plaice GST-A mRNA does not seem to suggest that bass GSTA functions as a housekeeping gene. This hypothesis is supported by a recent study by Hughes and Gallagher (2003) that suggested that bass GSTA mRNA expression may be altered by exposure to -naphthoflavone (BNF). Furthermore, other studies indicate that housekeeping genes such as

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41 glcyeraldehyde-3-dehydrogenase (GAPDH), B-globin, and human insulin growth factor (IGF) lack promoter elements (e.g. TATAA box and CAAT box), and most importantly, are devoid of response elements such as ARE, XRE, EPRE and GRE that may modulate gene transcription on exposure to certain chemical agents (Bird et al., 1987; Kim et al., 1991; McNulty and Toscano, 1995). In our study, genomic analysis revealed the presence of several putative promoter elements and putative response elements in the 5 flanking region of bass GSTA gene which further supports the hypothesis that bass GSTA is not a true housekeeping gene. Interestingly, the presence of several genomic clones with high identity to the plaice GST gene cluster indicates that a bass GST gene cluster is also present. Leaver et. al, proposed a possible role for plaice GSTA involves the detoxification of potentially deleterious fatty acid metabolites (1997). With addition of the complete nucleotide sequences of 1E, 1F, 1H bass clones and partial nucleotide sequences of 2F clone, it appears that the bass GST gene cluster is similar to the plaice GST gene cluster. Accordingly, the presence of such a GST gene cluster in a freshwater species phylogenetically distant from the plaice suggests conservation of an important function such as detoxification against lipid peroxidation. Interestingly, besides the plaice GST gene cluster, no other GST gene clusters have been reported in aquatic species. However other GST gene clusters have been reported in Anopheles gambiae (Ortelli et al., 2003), fruit fly (Sawicki et al., 2003), and the human GST alpha locus (Morel et al., 2002).

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42 Analysis of the 5 flanking region of the bass GSTA revealed a putative transcriptional start site (TSS), a putative enhancer elements including CAAT and TATAA boxes and several putative response elements (XRE, ARE, GRE and EPRE). The presence of a TATAA and CAAT box consensus elements (approximately -120 bp and -90 bp upstream, respectively, of the transcriptional start site) does not provide conclusive identity of these enhancer sequences, due to the fact that the majority of CAAT and TATAA boxes in eukaryotic genes are generally situated about -30 to -50 nucleotides upstream from the site of transcription (Nussinov, 1987; Li et al., 2000). Interestingly, Leaver et al. (1997) used primer extension to locate the transcriptional start site (72 bp upstream of the initiation codon) in plaice GSTA. Interestingly, we found a putative transcriptional start site 50 bp from the putative initiation codon for bass GSTA with identical sequences (CCGGCCCCCC) to the plaice transcriptional start site. However, the only way to determine the actual transcriptional start site of the bass GSTA gene will be to use such methods as SP1 protection or primer extension analysis. The presence of a putative EPRE and putative ARE located in the upstream region of bass GSTA (-5 bp and -180 bp, respectively) suggest that this gene may be inducible by phenolic antioxidants. This hypothesis is supported by mammalian studies that has shown phenolic antioxidants can induce GST via ARE (Hayes and Pulford, 1995). A previous study in our laboratory that showed an induction of GST activity followed by exposure to the antioxidant, ethoxyquin, in brown bullhead (Henson et al., 2001). In addition, a GRE-like element was

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43 identified in the upstream region of bass GSTA. Dexamethasone, a glucocorticoid-like inducer, has been shown to induce rodent GST via the GRE (Hayes and Pulford, 1995), but little evidence has been published about GRE-mediated GST induction in fish. The presence of putative XRE (located -855 bp from the putative transcription start site), correlates with our study that showed a slight induction of GSTA mRNA expression by BNF in bass (Hughes and Gallagher, 2003) which is consistent with a presence of a single XRE in the bass GSTA promoter. In summary, bass GSTA is the major GST isoenzyme in bass liver and rapidly catalyzes the GST-mediated conjugation of 4HNE. Based on sequence identity, catalytic activity and immunological cross-reactivity analysis, the bass GSTA isoenyzme differs from rodent GSTs and may be part of a novel GST family in fish. Furthermore, based on tissue expression analysis and promoter analysis, this GST isoenyzme does not appear to share any housekeeping gene characteristics, but is part of a bass GST gene cluster that is similar to the GST gene cluster observed in plaice. For plaice, this GST gene cluster functions in protection against lipid peroxidation and oxidative injury. However, further studies on the regulation of bass GSTA using a variety of model inducing agents that are targeted towards different response elements needs to be accomplished. Also of interest is the nature and identity of the second GST isoenyzme present in our HPLC analysis.

BIOGRAPHICAL SKETCH I received my Bachelor of Science at the University of Florida in microbiology and a minor in chemistry in 1999. I was awarded consecutive Johns Hopkins Fellowship Research Award in 1998 and 1999. I started my graduate program at the University of Florida in 2002. After my graduation, I will be recruited as regional director for a consultant firm in Los Angeles, California. 55